Here we propose a Functional In Vitro Pre-Screening Model that is a biomaterials testing adaptation of well- established cell culture systems developed over the last 15 years by J.S. Hong at the NIEHS in Research Triangle Park, NC. Our model provides a neuroelectrode tissue reaction testing regimen that (1) is comprised of a cell culture system that contains all of the brain cell types known to participate in the tissue reaction, and (2) enables the immunocytochemical measurements normally conducted in vivo as well as access to analysis of protein and gene expression, pharmacological intervention, and real-time monitoring. All of this is (3) performed under the rigidly controlled experimental conditions that are necessary to dissect the complicated mechanisms behind chronic neuroelectrode failure, thus allowing us to test specific hypotheses regarding the brain's response to a foreign body. The project has two specific aims. Specific Aim 1: Develop an in vitro cell culture based model of glial scarring by characterizing the cellular responses to a foreign body placed in a variety of CNS culture systems. This aim attempts to recreate glial scarring around a foreign body within four different rat cell culture systems: (1) an early embryonic midbrain culture system shown to be a good in vitro model of neuroinflammation observed in vivo, (2) a late embryonic cortical culture system, (3) an early postnatal cortical glia culture system, and (4) an aged cortical culture system. Each system's response to a representative biomaterial placed in culture will be assayed by immunocytochemistry, videomicroscopy, ELISA, Bio-Plex Luminex, and real time PCR for characteristic in vivo glial scarring behavior. We hope to, at minimum, reproduce microglial migration and attachment to the biomaterial and GFAP+ astrocyte process envelopment of the biomaterial. Specific Aim 2: Utilize the in vitro model to test the effect of serum factors, inflammatory cytokines, and microglia on the development of the glial scar. The best in vitro model developed in Aim 1 will be used to test the hypothesis that serum factors are essential for the development of a glial scar by adding various serum factors into serum-free media to rescue scar formation (glial scarring does not form in serum free media). ). Alternatively, we will add factors found in serum free media into serum containing media to test for inhibition of scarring. We will also test the hypothesis that inflammation is necessary for glial scar formation by stimulating the culture system with an immune challenge (LPS, IL-1B, and/or TNF-a administration). Blocking experiments will be conducted to determine the effect of IL-1B and TNF-a in gliosis. Finally, we will test the hypothesis that microglia are involved in glial scar formation by selectively removing microglia from the culture through treatment with leucine-methyl ester, a phagocyte toxin. PUBLIC HEALTH RELEVANCE A significant barrier to the human use of neuroprosthetic devices is the effect that the neuroinflammatory response has on degrading the fidelity of the signal recorded microelectrodes implanted in the CNS. This proposal describes a cell culture system comprised of all of the major cell types of the CNS that recapitulates many of the hallmarks of the brain tissue response to implanted materials. This system will be used to reductively characterize the tissue response to microelectrode materials, as well as screen for strategies intended to alleviate this response.